Construction of a Versatile, Programmable RNA-Binding Protein Using Designer PPR Proteins and Its Application for Splicing Control in Mammalian Cells.

RNAs play many essential roles in gene expression and are involved in various human diseases. Although genome editing technologies have been established, the engineering of sequence-specific RNA-binding proteins that manipulate particular cellular RNA molecules is immature, in contrast to nucleotide-based RNA manipulation technology, such as siRNA- and RNA-targeting CRISPR/Cas. Here, we demonstrate a versatile RNA manipulation technology using pentatricopeptide-repeat (PPR)-motif-containing proteins. First, we developed a rapid construction and evaluation method for PPR-based designer sequence-specific RNA-binding proteins. This system has enabled the steady construction of dozens of functional designer PPR proteins targeting long 18 nt RNA, which targets a single specific RNA in the mammalian transcriptome. Furthermore, the cellular functionality of the designer PPR proteins was first demonstrated by the control of alternative splicing of either a reporter gene or an endogenous CHK1 mRNA. Our results present a versatile protein-based RNA manipulation technology using PPR proteins that facilitates the understanding of unknown RNA functions and the creation of gene circuits and has potential for use in future therapeutics.

Enhancement of RNA annealing and strand displacement found in archaeal ribonuclease P proteins is conserved in Escherichia coli protein C5 and yeast protein Rpr2.

We analyzed modes of action of ribonuclease P (RNase P) proteins, C5 in Escherichia coli and Rpr2 in Saccharomyces cerevisiae, using a pair of complementary fluorescence-labeled oligoribonucleotides. Fluorescence resonance energy transfer-based assays revealed that RNA annealing and strand displacement activities found in archaeal RNase P proteins are prevalent in eubacterial (C5) and eukaryotic (Rpr2) RNase P proteins.

Pentatricopeptide repeat motifs in the processing enzyme PRORP1 in Arabidopsis thaliana play a crucial role in recognition of nucleotide bases at TψC loop in precursor tRNAs.

Proteinaceous RNase P (PRORP1) in Arabidopsis thaliana is an endoribonuclease that catalyzes hydrolysis to remove the 5'-leader sequence of precursor tRNAs (pre-tRNAs). PRORP1 is composed of pentatricopeptide repeat (PPR) motifs, a central linker region, and a metal nuclease domain, the NYN domain. The PPR motifs are single-stranded RNA-binding motifs that recognize bases in a modular fashion. To obtain insight into the mechanism by which the PPR motifs in PRORP1 recognize a target sequence in catalysis, N-terminal successive deletion mutants were overproduced in Escherichia coli, and the resulting proteins were characterized in terms of enzymatic activity using chloroplast pre-tRNA(Phe) as a substrate. Although Δ89, in which all PPR motifs are present, retained the pre-tRNA cleavage activity, Δ129 devoid of the first PPR motif (PPR1) had significantly reduced cleavage activity. Likewise, deletions of the second (PPR2) or third PPR (PPR3) motif abolished the cleavage activity, suggesting that PPR motifs play a crucial role in catalysis. A proposed recognition code for PPR motifs predicted that PPR2-PPR5 in PRORP1 recognize C, A/U, A, and U, respectively, whose sequence is in good agreement with C56-A57-A58-A59 in the TψC loop in pre-tRNA(Phe). Mutational analyses of nucleotide residues in the TψC loop as well as nucleotide-specifying residues (NSRs) in PPR motifs further suggested that PPR2 and PPR3 in PRORP1 favorably recognize nucleotide bases C56 and A57 at the TψC loop in pre-tRNA(Phe), respectively. This prediction and previous biochemical data were combined to construct a fitting model of tRNA onto PRORP1, showing that the mechanism by which PRORP1 recognizes pre-tRNAs appears to be distinct from that by bacterial RNase P.